Method of preparing pyridoxal phosphate



Patented Jan. 12, 1954 QFFIC No Drawing. Application January 20, 1951,

whims deneane triphe rhat are re It saeqbieet Q th ve ehie iov a create o xi sin pr fieqxtiem eh e ete pyridoxal phosphate. Other omeas'tnrbe a t 3 l bs ha' p osphate. While v'ai us. 299 as a nts be ea for. this our iv'e"havedoundthatthe' x finest co veniently effected by treating as aqutus' solution of pyridoxamin phosphate with activated carbon or manganese dioxide? Ryridoxamine phosphate; thestarting. material used in our process, having. the formula r can b cramm b phoephe yl ine rimer. a; w th. anhydrous iill snh rid acid as;- c'ordl'ng to the methoddescribed in J ;A. C. S; 210f-(1Q4'8L l Pursuant to a preferred embodiment of our. invention, we have found that anaqueoussolui tion of pyridoxamine phosphate prepared by dissolving the reaction product of pyridoxamine and anhydrous phosphoric acid-can. be; oxidized directly without first isolating. the pyridoxamine, phosphate. Thus, pyri-doxamine dihydrochlorida is firstreactedwith anhydrous phosphoric acid, and. the resulting reaction mixture containing pyridoxamine phosphate and excess phosphoric acid. is dissolved 'inwater, and this resulting solution is reacted with an oxidizing agent :to convert the. Dylridoxamine. phosphate to the desiredpyridoxal phosphate. For example, theaqueous 50111;; tion of the pyridoxamine-phosphoric acid reac'; tion mixture isi treated with an activated'char coal which adsorbs the pyridoxamine phosphate and oxidize this product to pyridoxal phosphate. Aiterseparating' the charcoaladsorbate, it Washer successively, with Water, dilute acid, and finally with water again to remove any n+ terminating inorganic compounds. The pyridoxal phosphate is then recovered from the charcoal adscrbate by elution with an aqueous; alkaline solution and the pyridoxal phosphate recovered ir'omthe resultingeluate. For this 'elutio'n we prefer touse dilute ammonia Water since this;

volatile base facilitates recovery of the pyridoxal phosphate from the eluate. Thus, the ammonium hydroxide eluate is evaporated under reduced pressure to obtain the ammonium salt of pyri doxal phosphate.

Alternatively, manganese dioxide can also be employdt'o oxidize the aqueous solution of the reaction product of pyridoxamide and phos phorie acid. In using manganese dioxide, this oxidizing agent is ad'ded directly to the aqueous solution of the reaction product previously ad justed to a pH of about 6 with alkali and the mixture warmed to about 50 C. forsufiicie'nt time to insure completion of the cxidation f'Th'e resulting reaction tissue is filtered to remove the precipitated manganese salt and th desired pyridoxial phosphate recovered from the filtrate. Inaccordance with a'furth'er embodiment of ourinventionlthe pyridoxal phosphate is readily:

separated from neutral or acidic aqueoussolu tions containing organic and inorganic phosphate, by adsorption oncharcoal. The interferi'ng substances can then be removed by Washing the resulting charcoaladsorbate toremove. said substances, and the pyridoxal phosphate recov ered in substantially. pure. form by elution of the washed adsorbate. Thus for example, the pyridoi ial phosphate is recovered from the filtrate described above by acidifying the filtratewith phosphoric acid; adsorbingfthe product from the acidifiedsolution on charcoalpreinoving the rei ll i icharqal ds b ts i't ch rted ad r ia was; was. d l id an fii y' with Water againto remove thev impurities elut -Q in g' the product from the washed charcoal adj sorbate withdilute ammonia water, andeva obtain: the almonium salt of pyrido ral plies;

phat. The following examples are presented. to illus;

mat s ecif embodimen Qt o r en ion,

3 Example 1 Seventy-five grams of phosphorus pentoxide was dissolved in 100 grams of 85% ortho phosphoric acid to give anhydrous phosphoric acid, according to the method described by Ferrel, Olcott and Frae'nkel-Conrat in J. Amer. Chem. Soc/70, 210 (1948). To grams of this anhydrous phosphoric acid, cooled to room temperature, was added 2 grams of pyridoxamine dihydrochloride. As the pyridoxamine dihydrochloride went into the solution there was a vigorous evolution of hydrogen chloride which was dissipated by vigorous stirring. When most of the gas has been evolved, the clear viscous solution was allowed to stand in a desiccator over phosphorus pentoxide for 3-6 days.

At the end of this time the reaction mixture was poured into about 300 grams of a mixture of crushed ice and water. The resulting colorless solution was treated with 35-40 grams of Darco G-60 (an activated charcoal), which adsorbed the phosphorylated amine. This adsorption is almost quantitative and can be followed by ultraviolet absorption spectrum. The charcoal adsorbate was filtered by suction and Washed well with water to remove the bulk of occluded inorganic compounds. It was then mixed with one or two volumes of Polycel, a purified wood cellulose fiber, to facilitate washing, and was placed in a chromatograph column.

This charcoal mixture was washed with dilute hydrochloric acid (0.5-1%) to remove the remainder of the inorganic substances. It was then washed with water to remove the hydrochloric acid. Both of these washings were carried out thoroughly, as none of the phosphorylated material was removed by them. Th charcoal adsorbent was washed with dilute ammonia water (0.54%) to elute the phosphorylated product. The eluate was vivid yellow in color due to the presence of the dissolved ammonium salt of pyridoxal phosphate. The phosphorylated amine had been oxidized by charcoal adsorbed oxygen to give the corresponding phosphorylated aldehyde. When about 700ml. of filtrate had been collected most of the dissolved product had been removed. This filtrate was concentrated to about 50100 ml. under reduced pressure and at a low temperature. It was then freeze-dried to give 1.5 g. of an amorphous brown hygroscopic powder containing the ammonium salt of pyridoxal phosphate. This substance showed strong codecarboxylase activity. The absorption spectrum of th ammonium salt of pyridoxal phosphate in aqueous solution mad alkaline with sodium hydroxide to pH 10.5 showed principal maxima at about 2300 A. and 3900 5.; a shoulder at 2625-2715 A; and a plateau at 3050-3200 A.

Example 2 Pyridoxamine dihydrochloride (2.5 g.) was dissolved in g. of the anhydrous phosphoric acid as described above with the accompanying evolution of hydrogen chloride. After standing 10-11 days at room temperature, the mixture was poured into about 200 ml. of ice and water. The acid was neutralized carefully with sodium hydroxide solution until the reaction mixture was about pH 6.

One gram of manganese dioxide was added and the mixture was heated at 60 C, for 20 minutes with frequent shaking. The manganese dioxide was replaced by a light-colored inorganic solid believed to be manganous phosphate, and the colorless solution became yellow-browri'due to the presence of the pyridoxal phosphate. The solution was cooled, filtered from th inorganic manganese salt, diluted with an equal volume of water and reacidified with phosphoric acid to Congo red. The pyridoxal phosphate was adsorbed on Darco G-60 as described in Example 1 and isolated in an identical manner. The yield of the freeze-dried residue was 2.6 grams. The absorption spectrum of the ammonium salt of pyridoxal phosphate in aqueous solution made alkaline with sodium hydroxide at pH 11.0 showed the principal maxima at about 2300 A. and 3900 A; a shoulder at 2650-2750 A. and a plateau at 2850-3100 A.

Example 3 'Twenty-five grams of pyridoxamine dihydrochloride was dissolved in 250 grams of the anhydrous phosphoric acid as described in Example 1 and converted to the ammonium salt of pyridoxal phosphate in an identical manner. Instead of concentrating the ammonia eluate to dryness, however, it was concentrated to a volume of about 1 liter. The concentrate was acidified to pH ca. 5 with dilute acetic acid, and to it was added a solution of 18.5 grams of calcium acetate in water. As a large amount of precipitate formed, it was necessary to add additional acetic acid to bring it back into solution. A small amount of amorphous insoluble material was removed by filtration through Super-cel, and the clear filtrate was diluted with three volumes of ethyl alcohol. The mixture was allowed to stand overnight in the refrigerator. The precipitate was centrifuged, washed with a mixture of alcohol and water, with a mixture of alcohol and ether, finally with ether, and dried in a vacuum oven at 40-45 C. The yield of the bright yellow calcium salt of pyridoxal 5-phosphoric acid was 29 grams (97%).

The absorption spectrum of the calcium salt of pyridoxal phosphate obtained by dissolving the product in an aqueous solution of mineral acid and neutralizing the resulting solution to a pH of 11 with sodium hydroxide showed principal maxima at about 2280 A., 3075 A., and 3900 A. and a shoulder at 2650-2750 A.

Ewample 4 Pyridoxamine dihydrochloride (5 grams) was converted to pyridoxal phosphate by the use of anhydrous phosphoric acid and manganese dioxide as described in Example 2. Instead of isolating the solid, however, the aqueous solution was treated with hydroxylamine hydrochloride, in a solution of about pH 2. The pyridoxal phosphate oxime crystallized slowly from solution. Yield 2.6 grams (50%) M. P. 218 C. dec. The absorption spectrum in aqueous solution of the oxime at its own pH showed principal maxima at about 2300 A 2865 A and 36005. These values compared closely with those obtained on analysis of the same product prepared by another method.

Example 5 0.108 gram of the calcium salt of pyridoxamine phosphate was dissolved in 25 ml. water containing enough dilute hydrochloric acid to effect solution. The resulting sodium salt of pyridoxamine phosphate obtained by taking 1 ml. of this solution and diluting to ml. with 0.1 N NaOH gave an absorption spectrum with principal maxima at 2475 A. and 3060 A.

Nine ml. of the starting solution was adjusted to pl-I 6 and to it was added 5.1 gram of mangancse dioxide. The mixture was heated at (ii -65 C. with frequent for 26445 minutes. The solution became yellow in color. It Was cooled and the inorganic salts filtered out. Because of the dilution due to washing, 2 nil. of the filtrate was diluted to 160 ml. with 0.1 N NaOH; the absorption spectrum of this solution had a shoulder at 2700 p ateau at 29% and maxima at 3900 in, show g that the pyridox amine phosphate had been converted to pyridoxal phosphate.

Various changes and modif'cations may be made in carrying out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are within the purview of the annexed claims, they are to be considered as part of our invention.

We claim:

1. The process for preparing pyridoXa-l phosphate which comprises contacting an acidic aqueous solution of pyridoxamine phosphate with activated charcoal.

2. The process for preparing pyridoxal phosphate which comprises reacting an acidic aqueous solution of pyridoxarnine phosphate with manganese dioxide.

3. The process which comprises contacting an acidic aqueous solution of pyridoxamine phosphate with activated charcoal, separating the resulting charcoal adsorbate, and eluting pyridoxal phosphate from said charcoal adsorbate with an aqueous alkaline solution.

4. The process which comprises contacting an acidic aqueous solution of pyridoxamine phosphate with activated charcoal, separating the resulting charcoal adsorbate, and eluting pyridoxal phosphate from said charcoal adsorbate with a1nmania water.

5. The process which comprises contacting an aqueous solution of pyridoxaininephosphate with activated charcoal, separating the resulting charcoal adsorbate, eluting pyridoxal phosphate from said charcoal adsorbate with ammonia water and recovering pyridoxal phosphate from said eluate.

6. The process which comprises reacting manganese dioxide with an acidic aqueous solution oi pyridoxamine phosphate, adsorbing the pyridoxal phosphate thus formed in the aqueous solution with activated charcoal, and eluting said pyridcxal phosphate from the resulting charcoal aolsorbate with ammonia water.

'2. The process which comprises intimately contacting an acidic solution of pyridoxamine phosphate with an oxidizing agent from the group consisting of activated charcoal and manganese dioxide to produce pyridcxal phosphate.

8. The process which comprises treating an aqueous solution of pyridoxamine phosphate at a pH of about 6 with manganese dioxide, and warming the resulting reaction mixture to about 60 C. to form pyridoxal phosphate.

STANTON A. HARRIS. ANDREW N. WILSON.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 2,497,732 Hoffman Feb. 14, 1950 OTHER REFERENCES Snell: J. Amer. Chem. Soc, vol 66 (1944), pp. 2082-2088. 

1. THE PROCESS FOR PREPARING PYRIDOXAL PHOSPHATE WHICH COMPRISES CONTACTING AN ACIDIC AQUEOUS SOLUTION OF PYRIDOXAMINE PHOSPHATE WITH ACTIVATED CHARCOAL. 